The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages

Atherosclerosis (AS) is a chronic and progressive inflammatory disease characterized by the accumulation of lipids and fibrous lesions in the large arterial intima. Mechanisms governing AS continue to draw extensive research interest. Among proposed mechanisms, the immuno-inflammation hypothesis has become accepted by most researchers[1‐3]. Macrophages are important in both the initiation and progression of AS[4, 5]. Macrophages can differentiate into two subtypes (M1 and M2) in response to micro-environmental changes. Considered the classical activated subtype, M1 exerts pro-inflammatory effects that promote the progression of AS. In contrast, the M2 activated subtype exerts anti-inflammatory effects and is regarded as a repressor of AS[6, 7].

Hyperhomocysteinemia (HHcy, abnormally high physiological levels of homocysteine, Hcy) is regarded as an independent risk factor in coronary heart disease, although the exact mechanism of how Hcy induces AS is unclear. Studies have shown that Hcy can promote AS occurrence and progression through oxidative stress damage, endothelial damage, and activation of inflammatory and immune responses[8, 9]. A positive correlation between HHcy, endothelial dysfunction, and accelerated AS has been demonstrated by using gene-induced and diet-induced HHcy animal models[10]. The results of some studies also reveal that Hcy could induce monocyte/macrophage localization in inflammatory lesions and exert pro-AS effects. Additionally, researchers have found that Hcy can influence the differentiation of macrophages both in vivo and in vitro[11]. However, it is unclear if the effect of Hcy on macrophage polarization and subtype conversion correlates to its pathogenic role in AS.

Macrophages are considered to be important immune effector cells. Recent studies have uncovered plasticity and heterogeneity in their differentiation pathways, as demonstrated by changes in cellular phenotype and physiology in response to micro-environmental stimuli. Mirroring the Th1/Th2 nomenclature, polarized macrophages are categorized into M1 and M2 subtypes. The classical subtype of activated macrophages, M1, can initiate adaptive immune responses and produce various pro-inflammatory cytokines. These biochemical responses are often involved in killing microorganisms and tumor cells. Moreover, M1 macrophages can promote atherosclerosis (AS). The alternative activated subtype, M2, also plays various roles, such as directing Th2 humoral response, killing parasites, repairing tissues, and producing anti-inflammatory cytokines. M2 macrophages are regarded as inhibitors of AS. However, it is noteworthy that the distinction between M1 and M2 macrophages may not be absolutely definitive; many researchers consider the M1 and M2 subtypes as opposing extremes of the macrophage spectrum[13].

The classical activation of M1 macrophages by interferon-γ (IFN-γ) and LPS creates cells that can produce various pro-inflammatory chemokines and cytokines such as IL-6 and TNF-α. M2 macrophages, usually activated by IL-4, produce anti-inflammatory cytokines such as IL-10. Aside from their differential expression of opposing effector molecules, inducible nitric oxide synthase (iNOS) is expressed more in M1 macrophages, whereas arginase-1 (Arg-1) and mannose membrane receptor (MMR) are expressed more in M2 macrophages, reflecting the different immunological roles of these two populations[7]. In this study, we chose IL-6, TNF-α, and iNOS as M1 macrophage markers, whose expression was induced by IFN-γ and LPS. We chose IL-10, Arg-1, and MMR as M2 macrophage markers, whose expression was induced by IL-4. These results are consistent with our previous work[12].

LPS, the primary glycolipid component found in the outer membranes of Gram-negative bacteria, can induce inflammatory responses, activate the complement system, and initiate the coagulation pathway[14]. Many studies have confirmed that in the presence of IL-1 or IFN-γ, the LPS-induced defense reaction and killing ability could be augmented. Conversely, the pro-inflammatory effect of IFN-γ can be enhanced by the presence of LPS[15, 16]. Because we had been unable to stimulate macrophage subtype polarization using Hcy alone (data not shown), we decided to use a combination of Hcy and LPS.

In the presence of LPS, Hcy led to up-regulated expression of M1 macrophage markers (IL-6, TNF-α, and iNOS); the optimal Hcy concentration was 50 μmol/L. However, Hcy and LPS elicited no significant effects on the expression of M2 macrophage markers (IL-10, Arg-1, and MMR). No correlation between the concentration of Hcy and the expression of marker genes was observed. This was possibly due to higher Hcy concentrations leading to more cell death. We also found that a combination of Hcy and LPS could transform M2 subtype into M1. This phenomenon may help to explain the pro-atherosclerotic effects of Hcy.

Many studies have shown that Hcy can act as a promoter of AS by activating inflammatory immune reactions. The immuno-inflammation hypothesis is a generally accepted explanation for the mechanism of AS, and macrophages are believed to be important in this process. The differentiation of macrophages into different sub-populations has become a new exploratory area in the initiation and progression of AS. Zhang et al. have demonstrated that Hyperhomocysteinemia (HHcy) could accelerate AS and enhance Ly-6C Monocyte/Macrophage accumulation in the lesions of Tg-hCBS apoE^−/−Cbs^−/− mice. They also found that monocyte/macrophage accumulation in the lesions of Ly-6C-positive mice correlated positively with Hcy levels. However, little is known about the influence of Hcy on the polarization of cultured macrophages[17]. In this study, we used a cell-based assay to demonstrate that Hcy could induce macrophage polarization into the pro-inflammatory phenotype, thereby providing further evidence in support of the model for Hcy-induced AS.

We investigated the synergistic effects of Hcy and LPS on the differentiation and conversion of RAW264.7 macrophages. The results showed that a combination of Hcy and LPS could induce macrophages to differentiate into the pro-inflammatory subtype. The differentiated anti-inflammatory subtype could also be converted to the pro-inflammatory subtype by combined treatment with Hcy and LPS. These findings provide new evidence for the role of Hcy in facilitating the progression of AS.